1. Field of the Invention
The present invention relates to anti-aggregatory agents for platelets, specifically oligopeptides which inhibit fibrinogen-platelet binding.
2. Background of the Related Art
The control and selective prevention of uncontrolled clotting is an important medicinal and clinical goal. The production of a blood clot in response to an injury can often become life-threatening when occurring at inopportune times and locations in the circulatory system.
Human blood platelets function to prevent bleeding by adhering to the surface of damaged blood vessels and then aggregating with one another. In pathological conditions, platelet aggregation and thrombus formation can be inappropriate, leading to occlusion of blood vessels, as occurs in heart attacks and strokes. These thrombotic diseases are some of the most common cause of death each year throughout the world. In addition, this same process is responsible for venous thrombosis, pulmonary embolism, and failure of vascular graft surgery, which are all serious medical problems affecting millions of patients; See, Green et al., "Anticoagulant Biotherapeutics for Heart Disease and Stroke" Anticoagulant, Spectrum Biotechnology Applications, Decision Resources, Inc. (Aug. 8, 1991).
A blood clot can obstruct a vessel, stop the supply of blood to the heart, limb or to an essential organ. Clots which become detached, called embolisms can flow through the circulatory system causing blockages at sites which are remote from the point of original injury and clot. If processes for blood clotting could be controlled, sufficient clotting can be obtained to allow healing of an injury without creating uncontrolled thrombi. Control of the clotting mechanism may be useful in preventing heart attacks due to reocclusion at the site of a clot, strokes, injuries due to embolisms and other clotting related injuries.
There are three related mechanisms involved in blood coagulation. The coagulation cascade leads to the generation of thrombin resulting in the conversion of fibrinogen to fibrin, a mesh-like structure that makes up a clot. The thrombolytic mechanism leads to clot dissolution by converting plasminogen into the clot-digesting enzyme plasmin. Platelet adhesion and aggregation, the third mechanism, contribute to clot formation by forming a cellular plug and by enhancing thrombin formation. Platelets may be activated by several different agonists such as adenosine diphosphate (ADP), epinephrine, collagen and thrombin that are released in the circulatory system in response to injury. These agonists have the ability to alter the surface glycoprotein GPIIb/IIIa receptors of platelets so that they can bind fibrinogen and other adhesive glycoproteins. Fibrinogen, a dimeric molecule binds to two platelets simultaneously and acts as a bridging molecule, producing an extensive lattice formation. The GPIIb/IIIa receptors are present at a very high density on the surface of platelets, each platelet containing approximately 40,000-50,000 such receptors.
The importance of the GPIIb/IIIa receptors in platelet aggregation is demonstrated by the failure of platelets to aggregate when taken from patients suffering from Glanzmann thrombasthenia, who lack the GPIIb/IIIa receptor, and the ability of monoclonal antibodies to the GPIIb/IIIa receptor to abolish platelet aggregation. Accordingly, non-immunogenic peptide-based inhibitors with high specificities would have tremendous therapeutic potential. These type of inhibitors could offer the prospect of rapid inhibition of platelet aggregation. In addition, they could be given repeatedly since they are small enough to be non-immunogenic. This is a substantial advantage over monoclonal antibodies which may be immunogenic.
The GPIIb/IIIa receptor is part of a superfamily of adhesion receptors, many of which share a common recognition site for peptides containing the short hydrophilic amino acid sequence arginine-glycine-aspartic acid (RGD). In fact, all proteins that bind to the platelets GPIIb/IIIa receptor, for example fibrinogen, yon Willebrand factor, fibronectin and vitronectin have RGD sequence in their cell binding domains. Synthetic peptides containing this sequence act as competitive, reversible inhibitors in assays of cellular adhesion. They compete directly for fibrinogen binding sites on platelets in a noncytotoxic manner.
Various linear RGD peptides have been synthesized for the purpose of preventing platelet aggregation, for example see, Samanen et. al., J. Med. Chem., 34, 3114-3125 (1991). Many of those synthetic linear RGD peptides are described in U.S. Pat. Nos. 5,047,380; 4,683,291; 4,578,079; 4,614,517; 4,792,525; 4,992,463; and in European Patent Publication EP 298 820 A1. U.S. Pat. No. 4,683,291 in addition to disclosing linear RGD peptides also discloses linear RGD polypeptides and branched RGD containing peptides.
Various cyclic peptides containing the RGD sequence have also been synthesized, as described in U.S. Pat. No. 5,023,233 and in foreign patent publications EP 425 212 A2; EP 410 537 A1; JP 2-174797 and GP 3,41915 A2, and PCT publication WO 90/02751.
Therefore, a purpose of the present invention is to control the blood coagulation process by providing peptide based agents which competitively inhibit fibrinogen-platelet binding.
Another purpose of this invention is to provide oligopeptides that cannot easily be degraded by hydrolytic enzymes, i.e. proteases and peptidases, in the circulatory system, and allow these oligopeptides to inhibit fibrinogenplatelet binding.
A further purpose of the present invention is the production of peptide based agents which are non-immunogenic and highly efficient in preventing fibrinogen-platelet binding so that these agents can serve as therapeutic drugs.